This invention relates to non-glycosylated human alpha-fetoprotein, its production in transgenic animals and plants, and uses thereof.
Alpha-fetoprotein (AFP) is a 70 kDa glycoprotein produced by the yolk sac and fetal liver. AFP is present in fetal serum at milligram levels, and, at birth, declines to the nanogram levels normally found in adult serum: increased levels of AFP in adult serum are indicative of a yolk sac tumor, a hepatoma, or of liver regeneration. The role of AFP during fetal development is not known, although it has been suggested that AFP protects a gestating fetus from a maternal immune attack or from the effects of maternal estrogen.
In vitro and in vivo experiments have shown that AFP has both cell growth-stimulatory and -inhibitory activities, depending upon the target cell, the relative concentration of AFP, and the presence of other cytokines and growth factors. For example, AFP can inhibit the growth of many types of tumor cells, and, in particular, inhibits estrogen-stimulated cell growth. Conversely, AFP stimulates the growth of normal embryonal fibroblasts. AFP has also been shown to have both immunosuppressive and immunoproliferative effects.
In order to exploit the various biological properties of AFP, it will be necessary to obtain sufficient quantities of this molecule in an efficient and cost-effective manner. Expression of AFP in recombinant systems has proven difficult because expression of wild-type AFP in eukaryotic cells generally results in the production of several isoforms due to differential glycosylation of AFP at a single asparagine residue (amino acid 233). Expression of AFP in prokaryotic systems typically produces misfolded and inactive protein that is aggregated and does not have the correct internal disulfide bonds. This misfolded AFP must be purified and refolded under conditions that allow for the formation of 16 disulfide bonds, a difficult and time-consuming process, which results in a very low overall yield of active, useful protein. Because the non-glycosylated form of AFP exhibits the same biological properties as the glycosylated form and allows for a more standardized, consistent product due to the lack of glycosylation variability, non-glycosylated AFP is preferred for commercial production. Therefore, there exists a need for an efficient method to produce non-glycosylated human AFP for commercial and therapeutic applications.